There are microphotometric methods for the measurement or sensing of morphological and biochemical properties of macroscopic groups of particles, cells, blood cells, tumorous cells and the like. These methods are based on the principle that, in a photometric apparatus, light which proceeds from each individual particle which is suspended in fluid and which is drawn or pushed through a measurement position, called the flow metering chamber, is detected using light-sensitive detectors such a photomultipliers or photocells and is measured according to its intensity and/or temporal distribution. The results of such measurements on individual cells are histograms which display pulse height distributions. Methods of this kind permit the measurement of up to several thousand cells per second. The principle area of application is in cell biology and, in particular, research into cancerous cells as well as experimental or quantitative cytology.
In addition, methods have been proposed for measuring the cells in the first instance individually or measuring their biochemical or morphological properties and, directly thereafter, sorting these cells on the basis of preselected criteria. Such sorting devices operate, for example, in accordance with the following principle:
After recording of the particle property within the flow metering chamber or in a fine free stream as the stream leaves a pore aperture, the suspension stream is broken down with the aid of a piezoelectric transducer into homogeneous fine droplets the diameters of which are in the range from a few microns to a few tens of microns. On the assumption that at a particular time after the photometric measurement a particular cell to be selected is present as a droplet in the suspension stream immediately before the dissociation of this particle-carrying fluid component, the fluid current is electrically charged. This means that the droplet, which becomes dissociated immediately thereafter, is also charged. The chain of charged and uncharged droplets is now conducted past an electrostatically charged pair of electrodes. At this point, only the charged droplets are deflected in the desired manner and are caught in a vessel. Devices are known for immediately depositing these cells in culture vessels in order, for example, to cultivate lines of cells which are ascribed to a respective particular selected cell. Such methods are used, for example, in genetic engineering.
In contrast to this "open" sorting system, closed arrangements have also been proposed (Zold or Wiecorek or Kamentsky). The arrangement described by Kamentsky makes use of a piezoelectric transducer for the temporally brief disturbance of a particle stream which, being surrounded by particle free solution, passes a measurement chamber. The disturbance of the particle stream, initiated as the result of the presence of a particle of interest is intended to lead to a situation in which the stream is mixed so that the pertinent particle passes into the enveloping current which is otherwise particle free, from which it is conducted into a separate trap.
The device described by Wiecorek et al makes use of a capillary duct system with, for example, a Y-shaped fork which is situated, in the direction of flow of the suspension, down stream of the measurement position. A microscopically small air valve is situated in each arm of the Y-shaped fork. Plungers which are secured at piezoelectric membranes and which incorporate small pistons open and close these valves in accordance with the sorting decision which is made on the basis of the individual measurement values for the cells. In the system which is operated with vacuum, the opening of a valve leads to the inflow of air into the pertinent arm of the duct system and, thus, to a reduction in the rate of flow. With the correct choice of the period of delay which elapses between the measurement of the cell characteristics and the opening of a valve, one can cause the cells which are of interest to flow into one branch and the cells which are not of interest to flow into the other branch of the capillary system. This sorting device can be operated with one or two oppositely acting valves.
A further sorting device, which was proposed by Zold, likewise operated with a closed fork capillary system, in which the individual duct branches were occluded to a greater or lesser extent by electrical discharge between microelectrodes and the "gas formation" caused thereby in order to deflect the cells which are of interest into the respectively desired duct.
The droplet sorter first discussed above comprises an open system in which microscopically small droplets (the size of droplets in fog) containing the cells must be conducted in free fall past electrically charged electrode plates charged to about 10-20 KV. It is very difficult, in the first place, to determine the time which elapses between the measurement, the charging of the droplet which then becomes dissociated from the fluid current and the time at which the droplet reaches the "target" area. The timing is complicated by the fact that the droplet is subject to movement by air currents and by friction with air. This time must, to a large extent, be determined empirically and does not necessarily remain constant. The quality of the sorting process (purity of the sample) must be determined by the rather troublesome procedure of once again measuring the "sorted" cells.
This technique also subjects the cells to a drastic pressure change in a short path. As the droplets are formed, the stream is ejected under pressure through a very small opening and thus go from a pressurized state to an unpressurized state in a very short distance, somewhat less than 1 mm. The cells are rather delicate and cannot survive this sudden depressurization which results in degassing and cell deformation.
A decisive further disadvantage of this arrangement is that a considerable proportion of the microscopically small droplets do not move in the desired manner into the collecting vessel. By reason of complicated charging conditions of the droplets on the one hand and of the parts of the apparatus on the other hand, and also because of the very small size of the droplets and their susceptibility to movement by air currents, a considerable proportion of the droplets move in an uncontrolled manner into the region in the vicinity of the instrument. This is of very great importance because the cells are stained, for example, with fluorescent dyes which are as a rule mutagenic and carcinogenic, or contain radioactive substances (e.g., 3H-thymidine). The aerosol which is formed with cells labeled in such a manner represents a real danger to the operating personnel who can become contaminated to a considerable extent by inhaling.
A further disadvantage of such open sorting systems is that they cannot be operated over a relatively long period of time under sterile conditions. However, it is important to be able to operate under sterile conditions because the sorted cells are frequently intended to be the starting material for new cell cultures.
The disadvantage of the closed arrangement proposed by Kamentsky is that, after each individual sorting process, which presupposes a respective disturbance of the central current, i.e., the mixing of the central stream with the enveloping current, a comparatively long period of time elapses until the central current containing the cell has once again become established and stabilized. In addition to this, the separation of cells which are of interest is, to a considerable extent, dependent upon chance: not all desired cells move far enough away from the central current stream, as a result of the piezoelectric disturbance, to be in a position to be eliminated with certainty from the periphery of the fluid current. The system, which has been known for almost twenty years, could not be implemented as a practical matter because it does not accomplish the sorting in such a way as to give usable results.
The arrangement proposed by Zold is not suitable for sorting cells with the objective of subjecting these to further cultivation. Cells are relatively fragile; they loose their vitality as a result of the required explosions. Moreover, the system is very costly because of the substantial discharges which must take place at short time intervals and at high frequency. The gases which are formed must also be removed quickly; gases would interfere with the sorting process because of their elasticity and the inclination of the system to natural oscillations.
In the arrangement of Wiecorek et al, the quantities of air which are introduced prove to be disadvantageous for similar reasons. The arrangement is accordingly relatively slow or sluggish and susceptible to oscillations because of the elasticity of the air. The microscopically small valves are difficult to produce, susceptible to breakdown and costly in terms of maintenance when it is remembered that, in the open condition, the air inlet slits have a width of only several microns to a few tens of microns. The sorting process requires constant monitoring and correcting of the fine adjustment of the width of the air inlet opening. The required supply of air renders sterile operation of the arrangement difficult.